Construction of a fully synthetic human scFv antibody library with CDR3 regions randomized by a split-mix-split method and its application

Journal of Biochemistry
Chang-Cheng YinXi-Yun Yan

Abstract

The randomization scheme of hypervariable region takes crucial role in construction of a synthetic antibody library. The codon bias and inevitable 'stop' codon of conventional 'NNK' and 'NNS' codons limit their applications. Here we report a split-mix-split DNA synthesis method that can control over the amino acid composition and distribution of randomized sequences effectually. A fully synthetic human antibody library with a diversity of 1.56 x 10(9) was successfully generated with complementarity determining region 3 (CDR3) randomized by this strategy. Sequencing analysis indicated that >60% of colonies had completely correct scFv genes and the amino acid composition and distribution were designed well in accordance. The utility was demonstrated by screening of scFv clones against BHL (anti-CD3 x anti-ovarian carcinoma bispecific antibody). These results proved the feasibility of the split-mix-split DNA randomization strategy in library construction and site-directed mutagenesis.

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Citations

Nov 27, 2015·New Biotechnology·Melissa BatonickMargaret M Kiss
Mar 4, 2010·Biotechnology and Bioengineering·Xin GeGeorge Georgiou
Jan 4, 2005·Journal of Immunological Methods·Naoya TsurushitaShankar Kumar
May 4, 2012·Organic & Biomolecular Chemistry·Tamil Selvi ArunachalamSabine Müller
May 10, 2020·Nucleic Acids Research·Laurens LindenburgFlorian Hollfelder

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